ABSTRACT
Three fluorescence signaling DNA enzymes (deoxyribozymes or DNAzymes) were successfully immobilized within a series of sol-gel-derived matrixes and used for sensing of various metal ions. The DNAzymes are designed such that binding of appropriate metal ions induces the formation of a catalytic site that cleaves a ribonucleotide linkage within a DNA substrate. A fluorophore (fluorescein) and a quencher (DABCYL, [4-(4-dimethylaminophenylazo)benzoic acid]) were placed on the two deoxythymidines flanking the ribonucleotide to allow the generation of fluorescence upon the catalytic cleavage at the RNA linkage. In general, all DNAzymes retained at least partial catalytic function when entrapped in either hydrophilic or hydrophobic silica-based materials, but displayed slower response times and lower overall signal changes relative to solution. Interestingly, it was determined that maximum sensitivity toward metal ions was obtained when DNAzymes were entrapped into composite materials containing approximately 40% of methyltrimethoxysilane (MTMS) and approximately 60% tetramethoxysilane (TMOS). Highly polar materials derived from sodium silicate, diglycerylsilane, or TMOS had relatively low signal enhancements, while materials with very high levels of MTMS showed significant leaching and low signal enhancements. Entrapment into the hybrid silica material also reduced signal interferences that were related to metal-induced quenching; such interferences were a significant problem for solution-based assays and for polar materials. Extension of the solid-phase DNAzyme assay toward a multiplexed assay format for metal detection is demonstrated, and shows that sol-gel technology can provide new opportunities for the development of DNAzyme-based biosensors.
Subject(s)
Biosensing Techniques/methods , DNA, Catalytic/chemistry , Metals/analysis , Catalysis , Fluorescein/chemistry , Fluorescence , Gels/chemistry , Ions/analysis , Sensitivity and Specificity , Silicon Dioxide/chemistry , Spectrometry, Fluorescence/methods , Surface Properties , Time Factors , p-Dimethylaminoazobenzene/analogs & derivatives , p-Dimethylaminoazobenzene/chemistrySubject(s)
DNA, Catalytic/chemistry , Fluorescein/chemistry , RNA/chemistry , Adenosine Monophosphate/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/pharmacology , Allosteric Regulation , Base Sequence , Catalysis/drug effects , DNA, Catalytic/chemical synthesis , DNA, Catalytic/metabolism , Fluorescence , Kinetics , Molecular Sequence Data , Nucleic Acid Conformation/drug effects , RNA/metabolism , Spectrometry, Fluorescence , p-Dimethylaminoazobenzene/analogs & derivatives , p-Dimethylaminoazobenzene/chemistryABSTRACT
pH6DZ1 is a synthetic deoxyribozyme that is able to couple catalysis with fluorescence signal generation. This deoxyribozyme has the ability to cleave itself at a lone ribonucleotide that is present between a pair of deoxyribothymidines, one modified with a fluorophore (fluorescein) and the other with a quencher (DABCYL). Herein, we report on the sequence truncation and secondary structure characterization of pH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicate that pH6DZ1 has a four-way, junction-like secondary structure comprised of four short duplexes, three hairpin loops, and three interhelical unpaired elements. Ten nucleotides, all located in two separate single-stranded regions, were identified as functionally indispensable nucleotides (complete loss of the catalytic function was obtained upon mutation). Nine nucleotides, most of which are also distributed in three single-stranded DNA elements, were identified as functionally vital nucleotides (at least a 1000-fold activity reduction was obtained upon mutation). Our study has shown that pH6DZ1 has a secondary structure that is more complex than those reported for other RNA-cleaving deoxyribozymes. The identification of functionally important nucleotides lays the foundation for future mechanistic studies on this DNAzyme. The elucidation of the secondary structure of pH6DZ1 should facilitate the future exploration of this unique DNAzyme for the development of DNAzyme-based biosensors.
Subject(s)
DNA, Catalytic/chemistry , DNA, Catalytic/metabolism , RNA/metabolism , Base Sequence , Catalysis , DNA Footprinting , DNA Methylation , DNA, Catalytic/genetics , Fluorescent Dyes/chemistry , Hydrogen-Ion Concentration , Metals/metabolism , Molecular Sequence Data , Mutation/genetics , Nucleic Acid Conformation , Plasmids/genetics , Protein Structure, Secondary , RNA/chemistryABSTRACT
DNA enzymes (deoxyribozymes or DNAzymes) are single-stranded DNA molecules with catalytic function. Previously, we isolated several RNA-cleaving deoxyribozymes with fluorescence-signaling properties. These special DNA molecules are capable of cleaving an RNA linkage embedded within a DNA sequence and flanked by a pair of deoxyribothymidines modified with a fluorophore (fluorescein) and a quencher (dabcyl). Here we report on the sequence truncation and secondary structure characterization of one such deoxyribozyme known as pH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicates that pH6DZ1 has a four-way junction-like secondary structure comprising four short duplexes, three hairpin loops, and three inter-helical unpaired elements.
